Purpose. To evaluate the efficacy and safety of epithelium-on corneal collagen cross-linking (CXL) using a multifactorial approach to achieve proper stromal riboflavin saturation. Methods. This non-randomized retrospective study comprised 61 eyes with progressive keratoconus treated with epithelium-on CXL. Chemical epithelial penetration enhancement , mechanical disruption of the superficial epithelium, and prolongation of the riboflavin-induction time until verification of stromal saturation were used before the UVA irradiation. Uncorrected and corrected distance visual acuity , refraction, corneal topography, and aberrometry were evaluated at baseline and at 1, 3, 6, and 12 months postoperative. Results. At 12-month, UDVA and CDVA improved significantly. None of the eyes lost lines of CDVA, while 27.4% of the eyes gained 2 or more lines. Mean spherical equivalent decreased by 0.74 D, and mean cylindrical reduction was 1.15 D. Irregularity index and asymmetry from Scheimpflug-based topography and Max-K at the location of cone from Placido-based topography showed a significant decrease. Higher-order-aberration data demonstrated a slight reduction in odd-order aberrations S 3, 5,7 (P = 0.04). Postoperative pain without other complications was recorded. Conclusion. Epithelium-on CXL with our novel protocol appeared to be safe and effective in the treatment of progressive keratoconus. Corneal collagen cross-linking (CXL) is a low-invasive treatment aimed to improve biomechanical stability in eyes with keratectasia –3. A phoIn CXL, riboflavin has a dual function acting both as a photosensitizer inducing the physical collagen cross-linking and as an absorber of the UVA irradiation, preventing damage to deeper ocular structures , 11. Pro The “standard CXL protocol” described by Wollensak and colleagues includes removal of the corneal epithelium in a diameter of 9 mm, followed by saturation of the corneal stroma using 0.1% isotonic riboflavin solution in 20% dextran . This pr In the current study, a multifactorial approach was utilized to enhance the riboflavin penetration by employing: (1) BAC-containing local medication; (2) hypotonic riboflavin solution without dextran; (3) increased riboflavin solution concentration; (4) mechanical disruption of the superficial epithelium (microabrasions); (5) prolongation of the riboflavin-induction time until objective verification of the stromal saturation is confirmed. By such an approach, this nonrandomized retrospective study aimed to evaluate the efficacy and safety of the epithelium-on CXL in treatment of progressive keratoconus. μm at the thinnest point measured by ultrasound pachymetry; (3) age ranging from 18 to 45 years; (4) Amsler-Krumeich keratoconus classification stage II to III. Exclusion criteria were: (1) history of herpes virus keratitis; (2) severe dry eye; (3) concurrent corneal infections; (4) previous ocular surgery; (5) hard contact lens wear ≤4 weeks before the baseline examination.In this retrospective, interventional case series, we reviewed the medical records of all patients with advanced progressive keratoconus who had 12-month observation time after the epithelium-on CXL treatment using our multifactorial approach. The treatment was performed at The Eye Department of the University Hospital North Norway, Tromsø, Norway, between September 15, 2009 and September 15, 2010. This study was approved by the regional ethics committee and adhered to the official ethical regulations for clinical research and the Tenets of the Declaration of Helsinki. Inclusion criteria included (1) documented progression of keratoconus during the last 12 months before treatment (increase of astigmatism or myopia by 1.00 D or increase in average SimK by 1.50 D); (2) minimum corneal thickness of no less than 400 Pre- and postoperative assessments consisted of slit lamp biomicroscopy, Scheimpflug-based corneal topo-/tomography , Placido disk-based topography and wavefront aberrometry , uncorrected (UDVA) and corrected (CDVA) distance visual acuities , ultrasound pachymetry , tonometry , and patients′ subjective evaluation of postoperative pain. The patients were examined at 1, 3, 6, and 12 months postoperative.without dextran  were applied alternating every 30 seconds, until the riboflavin saturation was verified by the slit-lamp inspection of the cornea and by the determination of the presence of riboflavin flare in the anterior chamber . It was followed by the application of two drops of local anesthetic proparacaine 0.5% , two drops of local antibiotic gentamycin 0.3%  followed by proparacaine again, one drop every minute for five minutes. All the drops were preserved by BAC , aiming to increase the epithelial permeability by chemically disrupting the tight junction proteins. A round Merocel sponge  of 5 mm in diameter was inserted into the conjunctival sac to provide a depot of riboflavin, and to produce microabrasions of the superficial epithelial layers caused by friction upon patient's blinking. Thereafter, two drops of proparacaine and two drops of 0.5% aqueous riboflavin solution  chamber . Under t2 within a circular diameter of 9 mm. During the irradiation, BSS was applied every three minutes, and proparacaine drops were added as needed.The cornea was subjected to UVA radiation for 30 minutes with a wavelength of 365 nm at a working distance of 5 cm. The UV-X lamp  provided an irradiance of 3 mW/cmAfter the UVA irradiation, two drops of atropine 1%  and 2 drops of gentamycin were applied. The cornea was protected with a soft bandage contact lens for 12–18 hours. Instructions were given to apply a mixture of 0.1% dexamethasone and 0.5% chloromycetin  eye drops four times daily for seven days, as well as to use artificial tears as needed.t-test with IBM SPSS Statistics v19.0 . P < 0.05 was considered statistically significant.All visual acuity values were recorded as Snellen values, converted to LogMAR for statistical analyses and then changed back to Snellen values for presentation purposes. Pre- and postoperative topography was analyzed using Precisio's irregularity index (IRI) as well as by measuring the central 5 mm using OPD indices. Statistical analysis was performed to compare the post-operative data with the preoperative data using the paired Sixty-one eyes of 53 patients fulfilled the inclusion and exclusion criteria. The mean age of the patients was 32 ± 10 years . 85% of the eyes (52 eyes) were from male patients.P < 0.05). At 12-month followup, none of the eyes lost lines of CDVA, while 27.4% of the eyes gained 2 or more lines and the safety index was 1.14. At the same time point, mean spherical equivalent refraction decreased by 0.74 D , while mean cylinder decreased by 1.15 D (P = 0.00).Figures P = 0.01), irregularity index (P = 0.01), and asymmetry (P = 0.01). The K-value from OPD did not significantly alter regarding Mean-K, while the Max-K showed a significant decrease (P = 0.02) at the location of the cone. P = 0.04) and total higher-order-aberrations (P = 0.05). Aberrometry data showed a reduction in odd-order- S 3,5,7 (P = 0.00) and thereafter a gradual increase to preoperative level at 12 months after the treatment (P = 0.15). Precisio-measured pachymetry in Patients reported moderate to severe postoperative pain during the first 4–12 hours, peeking at 4–6 hours after surgery.Discreet superficial epithelial layer damage could be observed on slit-lamp examination upon the verification of riboflavin saturation and after the CXL treatment. No serious complications were recorded during the follow-up period.Previous studies report conflicting results on the effects of epithelium-on CXL. While Pinelli and colleagues reported no significant difference in the analyzed parameters between epithelium-on and standard CXL , Wollensμm [μm before and after the corneal saturation with the 0.5% hypotonic riboflavin solution. Even though the clinical safety of CXL with hypotonic riboflavin solution has been documented [≈53 cm−1 for 0.1% isotonic Riboflavin solution to ≈42 cm−1 for 0.1% hypotonic riboflavin solution [P = 0.09) from 2738 ± 188 cells/mm2 to 2608 ± 311 cells/mm2.On the basis of previous studies reporting increased epithelial riboflavin permeability using hypotonic solution compared to isotonic solution , 27, 34,cumented , issues solution . To compsolution  and hencThe current protocol also employed mechanical scarification of the epithelial surface by creation of microabrasions caused by movement of a Merocel sponge over the corneal surface with patient's blinking. The amount of such micro-abrasions could obviously not be standardized, and it varied between cases. This may be the reason for a relatively large variation in saturation time (mentioned in the next paragraph).Finally, in addition to the chemical and mechanical enhancements, the current protocol demanded a slit-lamp verification of the stromal saturation before the UVA radiation . Our cliOur visual and refractive outcomes were comparable to other published CXL studies. In the current study there were no cases with a loss of ≥2 lines of CDVA, endothelial cell count did not change significantly, and there were no infections or other types of keratitis. Most of the patients reported pain peaking 4–6 hours after the treatment despite the mostly preserved epithelium. This may be explained by actinic-keratoconjunctivitis-like reaction caused by the UV exposure during the treatment and by the microabrasions caused on purpose, to enhance riboflavin penetration. K value and DSI  decreased significantly on the OPD II, Placido-based topography, as did the posterior elevation, irregularity index and asymmetry on the Precisio, Scheimpflug-based topography. However, our mean-K did not decrease in contrast to most other studies. In most of our cases, in addition to the Max-K decrease, a moderate increase in steepness on the opposite side of the cone occurred  from the surface (Detection of the demarcation line  after CXithelium . In 24 e surface , which iEpithelial absorption/filtering of the UVA light that could potentially lead to lesser energy delivered to riboflavin-saturated stroma has also been stated as an argument against the use of epithelium-on CXL. Different studies offer a variety of evaluation backgrounds of this matter. A study performed by Baiocci et al.  claimed Finally, even if the epithelium-on CXL leads to a shallower cross-linking compared to the epithelium-off, the density of collagen fibers in corneal stroma is much higher in the anterior portion where most of the collagen cross-links occur , 45. This retrospective study showed that epithelium-on CXL using our novel protocol appeared to be effective and safe in treating progressive keratoconus. The improvements in the visual, refractive, and topographic parameters in our patients indicate that epithelium-on CXL had sufficient effect to halt the progression of keratoconus and improve the corneal shape. A randomized controlled trial is warranted to verify that the effect of the current approach is comparable with the standard epithelium-off CXL. Furthermore, the combination of different enhancers, osmolality, and concentration of riboflavin should be explored to further improve the procedure.


Thickness changes of corneal sub-layers after phacoemulsification were investigated by spectral domain ultra-high resolution optical coherence tomography (UHR-OCT). The corneas (n = 26) of 26 age-related cataract surgery patients were studied. UHR-OCT was used to evaluate the thickness of Descemet’s Endothelium Complex (DEC), stroma, Bowman’s layer, epithelium, and full cornea at the center (CCT) before, one day after, and one week after surgery. Non-contact specular microscopy measured CCT, endothelial cell density, and morphology.The DEC, stroma, Bowman’s layer, and epithelium were visualized by UHR-OCT. Before surgery, the DEC in all cases appeared as a translucent space between two smooth opaque lines. One day after surgery, the posterior corneal surfaces in half of the eyes were wavy and irregular. Compared to the baseline, one day after surgery the thickness increases of the DEC, stroma, and CCT were 4.3 ± 2.6 µm, 25.5 ± 24.9 µm, and 32.1 ± 26.6 µm, respectively (P < 0.001). The morphology of the DEC and the CCT recovered to baseline one week after surgery (P > 0.05), but endothelial cell density was 8.7% less than at baseline. There were no significant changes in Bowman’s layer and epithelium after the operation. The pre-operative DEC thickness was positively correlated with the decreased endothelial cell density at 1 day after surgery .The DEC showed edematous thickening and different degrees of morphological changes after phacoemulsification. The DEC deformation and corneal edema recovered by one week after surgery, which indicated recovery of endothelial function. UHR-OCT is a useful tool to evaluate function of the DEC after phacoemulsification. Pre-operative DEC thickness may indicate the integrity of the endothelium and could be used for predicting endothelial cell loss after phacoemulsification.  The evolution of phacoemulsification surgery has resulted in a trend towards improved visual acuity with optimal safety and minimum invasiveness . HoweverSeveral methods have been used to assess endothelial function post-operatively: specular microscopy and in vivo confocal microscopy for endothelial morphology, fluorophotometric measurement of corneal barrier functions, and pachymetry for central corneal thickness (CCT) -9. HowevAll patients were candidates for phacoemulsification surgery and intraocular lens implantation at the Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China. The study was approved by the Wenzhou Medical College review board, and written informed consent was obtained from each patient. All patients were treated in accordance with the tenets of the Declaration of Helsinki. Age-related cataract was diagnosed on slit-lamp examination based on the opacity of the crystalline lens by the same examiner (ZC). For each patient, the cataract hardness was between II and IV according to the Emery and Little nuclear hardness classification . ExclusiAll subjects were tested pre-operatively as well as one day and one week after surgery. The cataract was diagnosed bilaterally for each subject, but only one eye of each subject was randomly selected for study. The measurements were executed between 10:00 am and 5:00 pm. During each visit, non-contact UHR-OCT, specular microscopy, and tonometry were performed in the selected eye of each subject by the same veteran examiner (ZC). The surgical procedure was performed by the same veteran surgeon (YZ). An Infiniti phacoemulsificator  was used. The surgical settings were shown in 2. Among the five patients for whom Duovisc was used, the lens nucleus of four subjects was graded III, and the nucleus of the other subject was graded IV. The mean phacoemulsification time in the Duovisc group was 58.8 sec, and 43.0 sec in the Iviz group. The bottle height was 100.0 cm. The mean phacoemulsification energy was 22.9 ± 8.9%. The mean size of corneal incision was 2.8 ± 0.4 mm. For seven patients, the corneal incision size was 2.2 mm, and for the other 19 patients it was 3.0 mm. No intracameral antibiotics or intracameral anesthesia was used. The surgical procedure was free of complications for all patients. A topical combination of steroids and antibiotics was given three times daily for 4 weeks as the post-operative treatment.Duovisc was used when the lens nucleus was hard, or corneal endothelial cell density was less than 2,000 cells/mmTo detect the sub-layers of the cornea, a custom-built high speed, spectral domain UHR-OCT instrument was used for this study. It was similar to the device used in previously published studies ,16. A suDuring imaging, the subjects were asked to sit in front of the UHR-OCT instrument and look straight ahead to an external central target. To image the structural characteristics and calculate the thickness of all corneal layers including the DEC layer, a 6.1 mm horizontal scan was taken when the light crossed the corneal apex. Custom software was used to process the raw data as described in a previous study . The refThe repeatability of measuring the thickness of the epithelium and Bowman’s layer has been validated by our previous studies using the same method ,19, and A non-contact specular microscope  with an autofocus was used to determine the CCT, the endothelial cell density, and cell shape . After tIntraocular pressure (IOP) was evaluated by a non-contact tonometer . No anesthesia was used during the examination. Three readings were taken and averaged.The Statistical Package for the Social Sciences  was used for data analysis. Data were presented as means ± standard deviations. Repeated measurement analysis of variance was used for overall statistical testing. A separate analysis was carried out for each layer of the cornea. Fisher’s Least Significant Difference (LSD) post hoc tests were used to determine if there were differences in thickness of total cornea and corneal sub-layers among all visits. Paired samples t-test was used to compare CCT between instruments at each visit. Changes in endothelial cell density by visit were tested using Wilcoxon test. Spearman correlations among different parameters were determined. P-values < 0.05 were considered statistically significant. Using a similar UHR-OCT instrument, Shousha et al.  found thThe study included 26 eyes of 26 age-related cataract patients . Pre-operatively, the DEC, stroma, Bowman’s layer, and epithelium were visualized in UHR-OCT images as bands separated by smooth opaque lines . Prior tThe thickness of the central DEC before phacoemulsification surgery was 12.7 ± 1.8 μm . One day2 . The visual acuity at 1 day post-operatively in the patients who showed deformation of the endothelium and who did not was 4.81 ± 0.25, and 4.89 ± 0.18, respectively (P = 0.060). The pre-operative IOP was 12.1 ± 2.3 mmHg, 12.8 ± 3.8 mmHg at 1 day after surgery, and 10.9 ± 2.8 mmHg after one week . The pre-operative DEC thickness was positively correlated with the decrease of endothelial cell density between pre-operative and 1 day post-operative values , corneal decompensation, bullous keratopathy, and loss of vision occurs [Similar to other studies -32, the n occurs . AlthougEstablishing the relationship between the thickness and en-face morphology of the DEC may help to better understand the corneal responses to surgical procedures that can cause subclinical wounding during phacoemulsification. The pre-operative DEC thickness was positively correlated with the decrease of endothelial cell density at 1 day after surgery. However, the relationship between endothelial cell density imaged by confocal microscopy at baseline and endothelial cell loss one day after surgery was not evident in the present study. Our data showed that morphological and thickness analysis of the DEC obtained by UHR-OCT provided a more precise indicator of endothelial cell damage than specular microscopy alone. The cross-sectional thickness of the DEC pre-operatively imaged by UHR-OCT may indicate the integrity of the endothelium. In turn, this information may be used for predicting the endothelial cell loss after phacoemulsification. For the patients who did not develop wave-like morphological changes, the DEC increased after surgery, but stromal and total corneal thicknesses did not change. This indicates that the DEC may be a more sensitive parameter than other thickness parameters. It may be not feasible for everyone to use UHR-OCT to image the DEC due to the high cost and limited availability of commercial UHR-OCT instruments. Measuring stromal or total corneal thickness may be valuable in roughly determining corneal response to surgery. Considering the high resolution and wide scan width, UHR-OCT may add an alternative modality for studying the DEC thickness and morphology; however, UHR-OCT may not replace specular microscopy for imaging the endothelial cells. The values for CCT estimated by UHR-OCT and specular microscopy were similar to one another. The thickness of epithelium in the present study, 47.4 ± 3.6 µm, is in agreement with previously reported values . HoweverThere were some limitations in this study. The follow-up time was only one week after surgery. Although we found that the corneal sub-layer thicknesses recovered post-operatively, other parameters such as corneal endothelial cell density did not return to baseline. It is possible that a longer follow-up period would have shown that the endothelial cell density tended to recover or possibly continued to decrease . A seconIn summary, one day after phacoemulsification in age-related cataract patients, the cornea became thickened principally due to stromal edema and to a lesser extent DEC edema. The DEC displayed different types of morphological changes. The complex sub-layer deformations and corneal edema recovered by one week after surgery. This indicated that endothelial cells, though less dense than before surgery, were able to recover the functional state and restore the normal thickness of cornea. The thicknesses of the epithelium and Bowman’s layer did not change during the early post-operative period. The cross-sectional thickness of the DEC pre-operatively imaged by UHR-OCT may indicate the integrity of the endothelium, which, in turn, may be used for predicting endothelial cell loss after phacoemulsification. Considering the high resolution and wide scan width, UHR-OCT may add an alternative modality for studying the DEC thickness and morphology, although UHR-OCT cannot replace specular microscopy in imaging the endothelial cells.


Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca L-serine, a non-essential amino acid, plays a critical role in neuronal development and function in the central nervous system. As well as a building block of proteins, L-serine is a crucial neurotrophic factor and a precursor for phosphatidyl-L-serine, L-cysteine, nucleotides, sphingolipids, and neurotransmitters such as D-serine and glycine 2+-activated K+ (KCa) channels present on the endothelium Ca channels on the endothelium and subsequent dilation of cerebral blood vessels. We also examined whether this vasodilating action would contribute to its neuroprotective effect on the brain after permanent middle cerebral artery occlusion (pMCAO) in rats.L-serine has also been shown to induce a reduction in mean arterial pressure via endothelium-dependent vasodilatation through the activation of apamin and charybdotoxin-sensitive CaTwo hundred and eighty six male Sprague-Dawley rats weighting 270±10 g were used in the present study . All procedures were in accordance with the institutional guidelines of Nantong University, which comply with international rules and policies. Ethics in accordance with the ARRIVE guidelines were followed in animal experiments and approved by the Animal Care and Use Committee of Nantong University, Nantong, China.L-serine, D-serine, charybdotoxin (ChTx), orthophthalaldehyde (OPA), aminooxyacetic acid (AOAA), apamin and strychnine hydrochloride were purchased from Sigma-Aldrich Corporation ; 2,3,5-triphenyltetrazolium chloride (TTC), methanol and N-acetyl-L-cysteine (NAC) from Merck Corporation ; and dimethylsulfoxide (DMSO) from Shenhe Chemical Reagent Co. Ltd. . All other chemicals were from Sinopharm Chemical Reagent Co. Ltd.  or Xilong Chemical Co. Ltd. .L-Serine was dissolved in normal saline, and used intraperitoneally (i.p.) at an interval of 12 h for 3 days. To determine the dose-dependent neuroprotective effect of L-serine, rats were randomly divided into 5 groups: sham-operated, vehicle, and L-serine 56 mg/kg, 168 mg/kg and 504 mg/kg. L-serine was initially used at 3 h after pMCAO. The vehicle group was treated with isodose saline. To investigate the time-window of L-serine efficacy, rats were randomly divided into 6 groups: vehicle and 5 L-serine treated groups. Onset of L-serine use  was respectively at 1 h, 3 h, 6 h, 12 h and 24 h after pMCAO.Ca (SKCa and IKCa) channels were blocked. In addition, strychnine , or DMSO as a control, were used 5 min ahead of L-serine. Strychnine was dissolved with saline containing 0.1% DMSO.To determine the underlying mechanisms of the neuroprotective effect exerted by L-serine on the ischemic brain, L-serine 168 mg/kg was intraperitoneally used 3 h after pMCAO. A combination of apamin and charybdotoxin (75 µg/kg of each) were slowly infused through the internal jugular vein over a 10-min period and 45 min before the L-serine injection to ensure that the small- and intermediate-conductance KFor the measurement of D-serine and L-serine concentrations, L-serine 168 mg/kg was used intraperitoneally 3 h after pMCAO, and the samples were obtained at different time points: 0 h , 0.5 h, 1 h, 2 h, 3 h, 6 h and 12 h after L-serine injection. For the observation of the influence of AOAA on L-serine efficacy, AOAA was intraperitoneally used at 25 mg/kg or 50 mg/kg 45 min ahead of the L-serine injection.Rats were fasted overnight with free access to water. All animals were anesthetized first with 2 mL enflurane and anesthetization was maintained with 10% chloral hydrate . Permanent MCAO was induced as reported previously Neurological function was evaluated at 72 h after pMCAO and scored on a 6-point scale C–VL)/2VC]×100, where VC is the volume of control hemisphere (left side), VL the volume of non-infarcted tissue in the lesioned hemisphere (right side).Cerebral infarct size was assessed using the TTC staining method 72 h after pMCAO, rats were anesthetized with 10% chloral hydrate  and perfused with 250 mL of normal saline and subsequently with 200 mL of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Rat brain was removed and then post-fixed for 24 h in the same fixative. The post-fixed brain tissue was cryo-protected in 20% then 30% sucrose in PBS. The brain tissue was then sectioned coronally 20 µm in thickness with a cryostat slicer . The sections from bregma −2.60 mm to −3.2 mm were prepared, mounted with neutral balata  and blotted onto slides, and then covered with a coverslip after Nissl staining, which was performed as reported previously After Nissl staining, neuronal cells in the cortex of the infarct area were identified under a high-magnification (×400) light microscope and counted by an investigator blinded to the grouping. For each rat, the mean number of neurons was obtained by examining three serial coronal sections. In each section, the number of neurons was averaged from three different vision fields of the cortex. Only intact neurons with a clearly defined cell body and nucleus were counted.We used the method described by Kiya et al. 2) were continuously monitored with a laser Doppler perfusion monitor  and the moorVMS-OXY™ system . Briefly, a sagittal skin incision about 1.5 cm long was made. Scanning probes  were carefully placed on the skull ipsilateral to the pMCAO under a stereotaxic device  with its center at 5 mm lateral to midline and 1 mm posterior to bregma, thus avoiding any large vessel. After successful operation, the rats were continually kept anesthetized to detect the influence of L-serine on CBF and SbtO2 in the ischemic cortex. The basal level of rCBF prior to pMCAO was assigned a value of 100%, and the rCBF levels under other conditions were calculated as percent values relative to the basal level. Mean values of SbtO2 were obtained by calculating the average level of 5 min monitoring before pMCAO, 30 min after pMCAO and 30 min after use of L-serine or vehicle.According to the literature Amsterdam, Netherlands) with a bolus injection of 2 ml ioversol 320 through tail vein at a rate of 0.3 ml/s at the start of scan . The layer with maximal ischemic area was selected for the comparison of CBF relative to the non-ischemic side of the brain. Relative CBF of the region of interest was calculated by the software of the CT system. Other three parameters of relative cerebral blood volume (CBV), mean transit time (MTT) and time to peak (TTP) were simultaneously obtained according to the literature To verify the change in rCBF detected with the laser Doppler perfusion monitor, cerebral perfusion imaging of rat brain was performed using computed tomograph (CT) perfusion imaging technique. The anesthetization of rats was maintained with 10% chloral hydrate . L-serine (168 mg/kg) or the equal volume of vehicle  was injected intraperitoneally 30 min after pMCAO. One hour later, non-enhanced CT scans were continuously carried out using the Philips Brilliance 64 CT scanner  was simultaneously monitored from the hip skin of the rat using a probe  During the measurement of rCBF, transcutaneous partial pressure of OData are presented as the mean ± standard error. Neurological deficit score data were compared using the Kruskal-Wallis rank-sum test and the Extended t-test for post hoc comparisons. Data from multiple groups were analyzed using one-way ANOVA and Scheffé’s test for post hoc comparisons. Data of cerebral L-serine and D-serine content were analyzed using two-way ANOVA followed by one-way ANOVA for the same treatments, and Student’s t-test for two different treatments at the same time point. Differences with P values less than 0.05 were considered statistically significant.2 were monitored; however, no notable influence was observed after the use of L-serine, apamin plus ChTx, or a combination of L-serine and apamin plus ChTx ; at 168 mg/kg, the neurological deficit score was significantly reduced by L-serine from the control value of 2.50±0.22 to 1.17±0.31 (p<0.01), and the infarct volume from 29.0±0.8% to 21.4±1.5% (p<0.01). A higher dose of L-serine (504 mg/kg) further reduced the neurological deficit score and infarct volume, but the reductions were not significantly different from those of the 168 mg/kg group , but that at 24 h after pMCAO did not (p>0.05). First use of L-serine at 12 h after pMCAO reduced only the neurological deficit score (p<0.01), but not the infarct volume  was used at 1 h, 3 h, 6 h, 12 h or 24 h after pMCAO and repeated every 12 h for 3 days. The neurological function and infarct volume were determined 72 h after pMCAO. As shown in  p>0.05, .p<0.01, To investigate the potential mechanism of the neuroprotective effect exerted by L-serine, we measured the CBF of the cortex with the laser Doppler perfusion monitor. L-serine treatment (168 mg/kg) did not change CBF of the cortex ipsilateral to the ischemic side in the sham-operated rat –a, but ep<0.05, p<0.01, see Similar increase in the rCBF of the ischemic side of rat brain after use of L-serine (168 mg/kg) was also detected by CT perfusion imaging technique p<0.05, . Moreove2 was reduced markedly in the rat after pMCAO  to block these channels in vivop<0.01, To investigate whether elevation of rCBF by L-serine was mediated through opening SKp>0.05, To exclude the possibility that the effect of L-serine on rCBF was mediated through activating glycine receptors, we used strychnine (i.p.), a glycine receptor antagonist, at a subconvulsant dose of 0.42 mg/kg p>0.05, p<0.05, To verify the mechanism underlying the neuroprotective effect of L-serine in rats, we carried out further studies using apamin and ChTx. The neurological deficit score in the apamin+ChTx group was a little higher than that of the vehicle group, but not significant p>0.05, . HoweverIschemic rats were injected with L-serine  3 h after MCAO. Normal rats received the same treatment. Samples of blood and ischemic cortex were harvested at 0.5 h, 1 h, 2 h, 3 h, 6 h and 12 h after L-serine injection.p<0.01), and then the concentration of L-serine decreased with time.As shown in p<0.01, p<0.01). However, L-serine levels in the brain of normal rats increased slowly; the peak level was reached at 3 h after L-serine injection , and then the concentration of L-serine decreased rapidly to the basal level  . However, the D-serine level in normal rats was not markedly increased after L-serine treatment  receptor co-agonist p>0.05, It has been reported that pyridoxal 5′-phosphate-dependent serine racemase converts L-serine into D-serine The present study found that L-serine used after pMCAO reduced the neurological deficit, infarct volume and cortical neuronal loss in a dose- and time-dependent manner, suggesting that L-serine can exert a neuroprotective effect on the brain in permanent cerebral ischemic rats. These results extend and are consistent with our previous observation on the effect of L-serine in ischemic/reperfused rat after MCAO To investigate the mechanisms underlying the neuroprotective effect of L-serine, rCBF was monitored before and after pMCAO and after use of L-serine. Surprisingly, we found that L-serine did not influence CBF of normal rats, but improved CBF and oxygen saturation of the ischemic cortex in pMCAO rats. This increase in the CBF of ischemic brain after L-serine treatment was also detected in the pMCAO mice. We suggest that the neuroprotective effect of L-serine may be mediated, at least partly, by improving CBF of the ischemic brain, a finding which has not been reported previously. This is a novel mechanism of the neuroprotective effect of L-serine apart from the glycine receptor-mediated mechanism we reported previously Because glycine receptors were involved in mediating the neuroprotective effect of L-serine, as we reported previously Ca and IKCa channels and hyperpolarization of the endothelial cells Ca and IKCa channels present on the endothelium of the rat MCA, and both IKCa and SKCa channels are involved in the hyperpolarizing responses of the rat MCA Ca and IKCa channel activity could diminish the basal tone of the cerebral parenchymal arterioles (PA), but not MCA, and the EDHF responsiveness of PA after ischemia/reperfusion may be preserved and have an important role in vasodilation under the conditions when NO synthase has been inhibited Ca and IKCa channels conduct the outward current in isolated PA endothelial cells, and blockade of SKCa and IKCa channels in PA will decrease the resting cortical CBF by ∼15% while opening the channels increased CBF by ∼40% Ca and IKCa channels without increasing the heart rate, and this effect is more pronounced in NO synthase-blunted rats than in control rats The vascular endothelium controls vessel tone by releasing nitric oxide (NO) and prostacyclin, as well as by a third pathway involving endothelium-derived hyperpolarizing factors (EDHF) Ca and IKCa channels. In order to confirm this, we carried out further studies. According to the method described by Mishra et al. Ca and IKCa channels. Surprisingly, the effect of L-serine enhancing blood flow in the ischemic cortex was blocked, suggesting that L-serine may induce vasodilation in ischemic brain tissue to elevate rCBF through SKCa and IKCa channels on the endothelial cells of cerebral blood vessels.For these reasons, we supposed that L-serine might improve CBF in the ischemic area through opening SKCa and IKCa channels blockers, the reducing effect of L-serine on the neurological deficit score and infarct volume was significantly reduced, suggesting that the neuroprotective effect of L-serine may be partly due to an increase in rCBF in the ischemic brain through a vasodilating action mediated by SKCa and IKCa channels.To clarify whether this vasodilating action contributes to the neuroprotective effect of L-serine, an additional experiment was carried out. With pretreatment of SKD-serine is an important co-agonist for NMDA receptors at the glycine-binding site and necessary for glutamatergic neurotransmission in many neurophysiological processes To exclude an involvement of D-serine-related mechanisms in the neuroprotective effect of L-serine, the concentrations of L-serine and D-serine in the ischemic cortex and blood were measured. After intraperitoneal injection of L-serine, the blood level increased rapidly; after a 1 h delay the cortical level of L-serine in the ischemic side also reached a peak that was several times higher than the control. However, L-serine levels in the cortex of normal rats were not significantly elevated. The blood-brain barrier may be more permeable to L-serine under ischemic conditions. Therefore, with the help of serine racemase, D-serine levels in the ischemic cortex was elevated after injection of L-serine, although the peak level was reached with an additional 4 h delay and the amplitude was smaller than that seen with L-serine. These results are consistent with previous reports Ca and IKCa channels on cerebral blood vessel endothelial cells. Activation of glycine receptors was not involved in this vasodilating action of L-serine, and neither was the neuroprotective effect of L-serine related to the conversion of L-serine into D-serine by serine racemase  Examples of changes in the cerebral blood flow of mice in vehicle and L-serine groups. (B) Mean values of rCBF before and after MCAO. **P<0.01, vs. vehicle group. Young male C57BL/6 J mice (25–30 g body weight) were obtained from the Laboratory Animal Center, Chinese Academy of Sciences . The animals were housed in groups (4–5 per cage) with food and water available ad libitum, in a temperature- and humidity-controlled animal facility with a 12-h light/dark cycle. Mice were anesthetized with 1.5% isoflurane in 30% O2/68.5% N2O mixture under spontaneous breathing. With the mouse in a prone position fixed in a head holder , the scalp was shaved and cut meticulously with a surgical knife to expose the thin skull over the bilateral cerebral and cerebellar hemispheres without causing brain trauma. The baseline CBF values were recorded for 5 minute by using the laser speckle technique . Briefly, a CCD camera  was positioned above the head, and a laser diode (785 nm) illuminated the intact skull surface to allow penetration of the laser in a diffuse manner through the brain. Speckle contrast (defined as the ratio of the SD of pixel intensity to the mean pixel intensity) was used to measure CBF as it is derived from the speckle visibility relative to the velocity of the light-scattering particles (blood). This was then converted to correlation time values, which are inversely and linearly proportional to the mean blood velocity. The mouse was then placed supine, and ischemia was induced. The rectal temperature was controlled at 37.0±0.5°C during surgery with a feedback regulated heating pad. After exposing the right carotid artery, a 5-0 silk suture was advanced into the internal carotid artery 12 mm from the lumen of the external carotid artery. The ipsilateral common carotid artery was occluded with a small surgical clip immediately after suture blockade. The animals underwent MCA occlusion permanently. L-serine of 268 mg/kg was intraperitoneally injected 30 min after pMCAO. Laser speckle perfusion images were obtained every 7.5 s, continuing throughout 90 min after pMCAO. CBF was analyzed two dimensionally over time by quantifying the area of infarct. The relative CBF was then reported as the ratio of baseline for each animal. Student’s t-test was used for the statistical analysis of results from vehicle group and L-serine treated group (6 mice for each group).(TIF)Click here for additional data file.


Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02–03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.Multidrug-resistant  Salmonella enterica serovar Typhimurium, which affects both humans and animals, is one of the most commonly reported NTS serovars Worldwide, nontyphoidal salmonellae (NTS) are a major cause of foodborne illness and commonly cause self-limiting gastroenteritis. The ubiquitous S. Typhimurium of a distinct MultiLocus Sequence Type (MLST), ST313, has emerged in several African countries and represents a major public health concern Bloodstream or focal infections caused by NTS are infrequent in developed countries S. Typhimurium ST313 is a clonal clade presently circulating in sub-Saharan Africa introduced by a common ancestry more than 50 years ago S. Typhimurium ST313 D23580 compared to other S. Typhimurium chromosomes et al.S. Typhimurium may be of clinical significance, increasing the invasiveness of the bacteria. Additionally, such partial selective genome degradation could potentially also lead to host adaptation to humans as described for host restricted serovars such as S. Typhi, S. Paratyphi A and S. Gallinarum S. Typhimurium ST313 D23580 has been extensively used in studies on host pathogen interaction to gain more knowledge about NTS infection and to elucidate the reason for the apparent increased pathogenicity of this clone S. Typhimurium ST313 might have adapted to occupy an ecological and immunological niche provided by HIV, malaria and malnutrition in Africa Next Generation Sequencing (NGS) studies have revealed that S. Typhimurium ST313 to invasive disease S. Typhimurium are conveniently studied in the mouse model, where S. Typhimurium causes a systemic typhoid-like infection. Additionally, various in vitro cell culture models have proven useful for the analysis of molecular mechanisms during host-pathogen interaction in S. enterica infections Several works have linked S. Typhimurium has been extensively studied over the last few years and genome studies, involving the application of cutting-edge NGS technologies, have been highly useful for a better understanding of differences between strains. So far, genes encoded from Salmonella pathogenicity islands (SPIs) have been reported as the most significantly factors contributing to host cell interactions The pathogenesis triggered by st313-td (924 pb) through comparative genomics. This gene, of unknown function and unique to S. Typhimurium ST313 and S. Dublin, is harboured in a putative uncharacterized genomic island termed ST313-GI of ∼18 kb in the S. Typhimurium ST313 genomes. Among the genes of unknown function contained in ST1313-GI, st313-td, was the only one present in both, ST313 and Dublin chromosomes In a recent work we have identified a putative virulence associated gene designated st313-td from S. Typhimurium ST313 in virulence. In addition, the presence, frequency as well as the expression of the putative virulence gene was estimated in strains of S. Typhimurium and S. Dublin from a global strain collection. Furthermore, the genomic structure of the genomic island ST313-GI in S. Typhimurium ST313 was revealed.The aim of this study was to assess the role of the gene The clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. Mice infection studies were performed with permission to John Elmerdahl Olsen from the Danish Animal Experiments Inspectorate, license number 2009/561–1675.(www.ncbi.nlm.nih.gov) and the Uniprot database (www.uniprot.org) were used to determine the putative role of the ST313-GI encoded proteins. The analysis of the GC content and map construction of ST313-GI were performed with the Clone Manager Suite 9.0 software.Open reading frames (ORFs) of the genomic island ST313-GI were determined using the gene finder named Prodigal S. Typhimurium and 50 S. Dublin isolates from 10 countries were analysed for the presence of the gene st313-td (S. Typhimurium strains were associated to bacteraemia (isolated from blood specimens) whereas the remaining isolates (72.2%) originated from other sources such as food, stools etc.From a global strain collection maintained at the National Food Institute of the Technical University of Denmark (DTU-Food), a total of 295 st313-td . Eighty-S. Typhimurium 02–03/002 was used in all the experiments conducted in this study representing the clone S. Typhimurium ST313. This strain was isolated from a bacteremic child in the region of Lwiro (Democtratic Republic of Congo) in 2002–2003 S. Typhimurium non-ST313 strains, 4/74 and 14028, were used as controls in the different assays. The virulence properties of 4/74 and 14028 have been previously described The isolates were inoculated in Luria-Bertani broth or plates (LB) (Oxoid) and incubated for 16 h at 37°C. In addition to these conditions, broths were shacked at 200 rpm during incubation. If necessary, the LB medium was supplemented with antimicrobials at the following concentrations: kanamycin (50 µg/mL), apramycin (75 µg/mL), gentamicin (25 µg/mL) (Sigma).st313-td was evaluated by PCR using the primers st313-td-F and st313-td-R (TAG Copenhagen A/S) (The presence of the gene gen A/S) . The folS. Typhimurium strains collected in Belarus  S. Typhimurium 2006–130 and S. Typhimurium 2007–024, from stools) S. Typhimurium B1 and S. Typhimurium D12, from water) st313-td, were sequenced by Illumina GAIIx genome analyzer . The raw reads were subsequently submitted to the European Nucleotide Archive (www.ebi.ac.uk/ena/) under accession no. ERP004195. The raw reads can be accessed freely from the following link; http://www.ebi.ac.uk/ena/data/view/PRJEB4869. Assembly was performed by using the pipeline available on the Center for Genomic Epidemiology (www.genomicepidemiology.org). The latter is based on Velvet; algorithms for de novo short reads assembly Six http://mlst.ucc.ie/.MultiLocus Sequence Typing (MLST) was performed on the six isolates according to MLST Databases: S. Typhimurium ST313 D23580 genome as a reference. A SNP tree showing the divergence/relatedness of the Salmonella Typhimurium strains carrying st313-td, assigned to different ST types is shown in SNPs were identified as previously described 600nm  = 0.4±0.01). RNA was isolated from 1.5 ml aliquots of overnight cultures by mechanical disruption with the FastPrep system  and by using the RNeasy mini kit (Qiagen). Quantity and quality of total RNA was determined with the NanoDrop 1000 spectrophotometer (Thermo Scientific). The RNA was DNase treated with the TURBO™ DNase and reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the supplier's recommendations. The qPCR was done with the Maxima SYBR Green/Rox qPCR Master Mix and gene specific oligonucleotides (Table ) in a MxPro3000 cycler. The gene rsmCBacteria were grown to logarithmic phase in LB (ODThe wild type strains (WT) and plasmids used for the cloning and mutagenesis experiments as well as the mutated isolates constructed are listed in st313-td was performed using Lambda Red Mediated Recombination as previously described by Datsenko and Wanner st313-td. Since the WT strain was resistant to ampicillin, a derivative conferring gentamicin resistance (encoded by the gene aac(3)-Id) of the helper plasmid pKD46 was used aac(6′)-Iaa (conferring resistance to Kanamycin). The apramycin resistance-encoding gen (aac(3)IV) was amplified from the plasmid pUO9090 . The resulting construct, Δst313-td, was verified by PCR for the absence of the gene st313-td.The mutagenesis of 02–03/002 for the gene st313-td obtained from 02-03/002 was cloned into the restriction endonuclease site ScaI-PstI of the vector pACY177 st313-td. The presence of the insert was confirmed by PCR and restriction endonuclease assays. The restriction deficient strain Escherichia coli KP1274 was used as primary recipient for plasmids st313-td are listed in The PCR amplicon corresponding to the gene st313-td competent cells in order to obtain the complemented strain 02-03/002-C.After being recovered from KP1274 competent cells, the plasmid pAHF4 was also electroporated into ΔThe WT and the mutated strains  were asset al. 3 bacteria of each strain. The CFU and ratio between WT and mutated isolates were enumerated by plating as described Infections of female six-week old C57/BL6 mice were performed as described by Jelsback st313-td in the intracellular survival and replication within mouse macrophages was investigated using J774.1 cells; a mouse monocyte-derived macrophage-like cell line as previously described ssaV were used as positive and negative control for the infection assays, respectively. The latter is deficient in ssaV, a structural component of the SPI2-encoded T3SS The role of the gene TM-I, Earles, 25 mM HEPES (Gibco) supplemented with 10% (v/v) heat-inactivated FBS and 25 µg/mL gentamicin. Cells were incubated in a humidified 37°C, 5% CO2 incubator. The bacteria were grown in LB to a stationary phase, harvested at 8000 rpm for 5 min and resuspendeded in 0.9% (w/v) NaCl. Complement-opsonisation of the bacteria prior to infection, if used, was performed by mixing the bacteria with mouse serum followed by incubation at 37°C for 20 min. Bacteria were added at a multiplicity of infection of 10∶1 (bacteria complement-opsonized) or 50∶1 (bacteria not opsonized). Monolayers were centrifuged at 300 rpm for 5 min immediately after addition of the bacteria followed by incubation for 25 min at 37°C, 5% CO2. Enumerations of the bacteria in the inoculum were verified by plating onto LB agar plates (counted before infection). After infection, the media was removed and monolayers were washed twice with 0.9% NaCl. At this point (defined as time 0 h) fresh media containing 250 µg/mL amikacin was added to kill the extracellular bacteria. The plates were incubated for 1 h at 37°C, 5% CO2 before new media supplemented with 100 µg/mL amikacin was added for the remaining part of the experiment. This high concentration was used to ensure the killing of extracellular bacteria . To enumerate the bacteria, cells were washed twice with 0.9% NaCl, subsequently lysed in 1 ml 0.1% Triton X-100 (v/v). The viable intracellular bacteria were enumerated by colony counts of lysate dilutions plated on LB agar plates. For intracellular survival, bacteria were enumerated at t = 1 h , and t = 20 h . When opsonization was not performed, only survival after 1 h post-uptake (t = 1 h) was measured. Values determined at t = 20 h were expressed relatively to CFU for the WT determined at T1. The experiments were carried out in quadruplicates.Briefly, macrophage cells were cultured in RPMI+GlutaMAXCytotoxcity was assessed by measuring the release of cytosolic lactate dehydrogenase (LDH) into the supernatants as previously described st313-td and the invasion of the human epithelium was analysed using Int-407 cells . The isolate 4/74 and the isogenic strain invH201::TnphoAinvH gene, reducing its rate of invasion with regard to the WT strain.The association between the gene TM-I, Earles, 25 mM HEPES (Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum  and 25 µg/mL gentamicin to prevent bacterial contamination. The cells were grown statically in a humidified 37°C, 5% CO2 incubator. Twenty-four hours prior to infection, the Int-407 cells were seeded in 24-well plates in a concentration of 5×105 cells per well and left under the mentioned conditions. The bacteria were grown for 16 h, 200 rpm, 37°C in LB and sub-cultured (∼107 CFU/mL) into fresh LB and incubated for additional 3 h. At this point, when the bacteria reached an OD600∼0.5, the cultures were centrifuged at 6000 rpm for 5 min. The bacteria were resuspended to OD600 = 1.0 (1×109 bacteria per ml) in 0.9% NaCl and added to monolayers at a multiplicity of infection of 100∶1 (bacteria to eukaryotic cell). Equal inoculum counts were verified by plating on LB agar plates (counts before infection). After 1 h of infection at 37°C, 5% CO2, the media was removed and monolayers were washed twice with 0.9% NaCl. At this point, fresh media containing 250 µg/mL amikacin was added to kill extracellular bacteria. The plates were incubated for further 2 h at 37°C, 5% CO2. To enumerate invaded bacteria (t = 2 h), cells were washed twice with 0.9% NaCl and lysed in 1 ml 0.1% Triton X-100 (v/v). The viable intracellular bacteria were enumerated by colony counts of lysate dilutions plated on LB agar. The experiments were performed in quadruplicates.The epithelial cells were cultivated in “Eagle Minimum Essential medium” MEM+ GlutaMAXStatistical significance of the differences between the strains was determined using the GraphPad Prism 6.1 program. In all the assays, the statistical significance  was determined by one-way ANOVA analysis with pair-wise comparison of means by using Tukey's multiple comparison test.st313-td is located within a potential pathogenicity island ST313-GI in S. Typhimurium ST313 in silico studies. The sequence of ST313-GI to determine its structure has been extracted from the publicly available and annotated genome of D23580 (accession no. FN424405.1). It is to note that the gene island is not contained within any of the five prophages described in the mentioned genome. ST313-GI, of 17836 bp, is flanked by tRNA genes encoding for threonine (Thr) at the 3′end, and asparagine (Asn) and threonine, at the 5′ end, respectively. Such integration between tRNA encoding genes has typically been described for genomic islands in bacterial chromosomes Salmonella phages and other genomic islands st313-td. The remaining 21 ORFs of ST313-GI encode phage-related functions, such as replication, integration, excision etc.  out of the 82 S. Typhimurium strains from blood specimens exhibited the amplicon corresponding to the gene st313-td (S. Typhimurium strains collected from other sources and only 45 (21.1%) displayed the band expected for st313-td. The latter included 32 and seven isolates originated from stools in Belarus st313-td gene was absent in the remaining 168 (78.9%) non-blood S. Typhimurium isolates, including those collected from animals (n = 73) and food (n = 9) (S. Dublin strains from human (n = 25) and cattle (n = 25) harboured the st313-td gene  . On the -td gene .Thus, reS. Typhimurium strains carrying st313-td belonged to the lineage ST313. Thus, six non-invasive S. Typhimurium isolates representing a wide geographical distribution and positive for the gene st313-td were subjected to whole genome sequencing to assign a specific MLST type. None of the six strains from Belarus, China and Nepal belonged to ST313. Thus, the four non-blood isolates from Belarus and Nepal were assigned to the MLST ST328, while the remaining two non-blood isolates from China belonged to ST19 which is one of the predominant S. Typhimurium genotype in the MLST database (http://mlst.ucc.ie/). MLST ST313 differs from ST19 at only one MLST locus (sucA), which in turns differs from ST328 at the locus aroCS. Typhimurium carrying st313-td is illustrated in Figure S2. The analysis revealed that the strains clustered in two separate groups. One group included strains of African origin (assigned to ST313) and the second group contained strains from Asia and Europe (assigned to ST19 or ST328). These two groups of strains could either have a common st313-td ancestor or the gene could have been transferred independently into the two groups from another source.We assessed whether the non-blood st313-td gene was actually expressed in the different isolates where we had detected the gene, RT-qPCR was performed on a selection of strains. The collection included; two strains of S. Dublin  and 12 strains of S. Typhimurium  . In these genomes, only a fragment of ∼6.8 kb of ST313-GI carrying st313-td was detected To study whether st313-td gene and analysed by whole genome sequencing in the present work, only a fraction of ST313-GI was present. The ST313-GI fragments differed in size between the strains tested: a fragment of 1467 bp was detected in strains from Belarus, of 4245 bp in strains from China, and of 11317 bp in strains from Nepal. Therefore, the presence of the entire ST313-GI seems to be restricted to the S. Typhimurium ST313 lineage circulating in sub-Saharan Africa.In the six genomes of isolates from Belarus (n = 2), China (n = 2) and Nepal (n = 2) harbouring the st313-td plays a role in virulence, the mutant strain, Δst313-td was tested in competition with the wild type strain in a mouse model of systemic infection. Interestingly, the deletion of st313-td decreased virulence in C57/BL6 mice    versus the WT 4/74 was slightly increased, yet not significantly  (In order to investigate whether  <0.001) . Introdue >0.05) . Besidese >0.05) .et al.Virulence testing was based on competition for success between WT and mutant in the same animal, as suggested by Beuzon S. Typhimurium in the mouse model of systemic disease was reduced in the absence of st313-td. In order to study what might have caused this decrease, the ability of the mutant and complemented strains to infect and survive inside J774 macrophages was analysed. In the macrophage experiments where the strains were complement-opsonized, the WT 02–03/002 showed significantly  less intracellular counts at t = 1 h post-infection than the mutant isolate Δst313-td (Virulence of st313-td . On the st313-td was significantly (3.5 fold) reduced  compared to the WT 02–03/002. Interestingly, at this time point, the WT 02–03/002 and the complemented, 02-03/002-C, strains showed a similar net replication of ∼7 significantly higher than observed for the isolates 4/74 and 4/74-C  was observed between the WT 02/03–002 and the mutant Δst313-td at 1 hour post challenge  that the phenotype of the WT 02/03–002 and 4/74 was not affected by the introduction of the plasmid pACY177 used for complementation (data not shown).Theoretically, since cytotoxic effects results in exposure to the antibiotic in such cell culture experiments, a strain with increased multiplication ability could be masked by an increase in cytotoxicity. However, the mutated strains did not differ significantly from the WT in cytotoxicity 20 h post infection  . Same reSalmonella virulence. This aspect of infection was bypassed in our mice experiments, since we used intra peritoneal challenge. We therefore tested the role of st313-td in invasion in an epithelial cell infection model. The gene st313-td was not involved in invasion of the human epithelial INT-407 cells  compared to the WT 4/74 however, its CI increased by 50%. The data derived from mice experiments showed that the mutant, Δst313-td, is significantly less pathogenic than the WT, suggesting that st313-td contributes to virulence during the systemic infection in mice. The results from the mice experiments performed with the WT 4/74 and the mutant 4/74-C must be regarded as inconclusive, but probably other factors (apart from this encoded by st313-td) not present in WT 4/74 may be required to lead to an increase in virulence.Interestingly, an evident role of st313-td. These assays were considered relevant because infection and intracellular survival in macrophages is required for full virulence and infection of the intestinal epithelial layer is the first critical step in Salmonella virulence st313-td caused an increase on numbers of intracellular bacteria in macrophages at t = 1 h post-infection, but only in experiments where opsonisation was performed. This indicates that st313-td influences the uptake and/or early survival in macrophages but only in activated cells. Notably, in a previous study it was demonstrated that S. Typhimurium D23580, assigned to ST313 and carrying st313-td requires high levels of complement for antibody-mediated bactericidal activity compared with S. Typhimurium LT2 st313-td might play a role in virulence by either decreasing the binding of the bacteria to antibodies causing less uptake in macrophages or by diminishing the susceptibility to complement mediated lysis. Further studies are required to confirm this hypothesis.Additionally, infection of mice macrophages and human epithelial cell lines were performed to further detail the virulence enhancing role of st313-td caused a significantly decreased intracellular survival. It is well known that Salmonella are facultative intracellular bacteria and their ability to survive within cells is essential for virulence st313-td appears to encode a factor that favours the persistence of intracellular Salmonella. This trait may explain the virulence enhancing effect in the mice studies. These data indicate that the virulence enhancing role of st313-td appears to be dual and related both to a decreased uptake of bacteria in phagocytic cells and a better survival at late time points. Both these phenotypes may add to a higher ability to cause systemic infection, which is a hall mark of ST313. Results of cell culture infection can be influenced by levels of toxicity, since bacteria that kill the macrophages subject themselves to killing by antibiotics in the media. It is therefore important to note that strains tested in the macrophage infections showed similar cytotoxicity levels at t = 20 h.Once inside the macrophages, lack of st313-td did not influence invasion of epithelial cells. Thus the function of the gene product seems to be specific for phagocytic cells, and hence only relevant for systemic infection.Contrary to the results using macrophages, deletion of S. Typhimurium ST313 is essential during the conditions used for mutagenesis.Attempts to delete the whole genomic island ST313-GI were performed several times as part of the current study with no success. These data suggest that the carriage of ST313-GI in st313-td to isolates of S. Typhimurium from cases of bacteraemia was observed by PCR and furthermore, S. Dublin, which is a serovar highly invasive in humans st313-td. The gene was also shown to be expressed in a collection of both S. Dublin and S. Typhimurium strains. These data support a potential role of st313-td in systemic salmonellosis caused by these serovars.A strong association of the gene S. Dublin isolates proved to harbour the gene, this serovar is a likely ancestor of the gene st313-td, subsequently transferred to S. Typhimurium. Further analysis should be performed in order to confirm this speculation and to investigate whether it plays a role in S. Dublin virulence.Taking into account that all the st313-td in a genetic mobile element, the genomic island ST313-GI, supports our hypothesis that the gene might have been transferred by horizontal gene transfer to S. Typhimurium. It is noteworthy that Wain et al. S. Typhimurium ST313 likely emerged from a pathogen which was already adapted to an animal host, causing diarrhoea in healthy humans and invasive disease in HIV-infected patients as a result of their immune deficiency.The presence of st313-td is also supported by the fact that six strains isolated from human stools in Belarus and China, and from water in Nepal assigned to other ST types  than ST313 also carried the gene. However, in contrast to the ST313 strains, the six non-ST313 isolates analysed did not harbour the entire ST313-GI and only small portions of the genomic island matched the genomes of these strains. Therefore, contrary to the situation observed for the single gene st313-td, the genomic island ST313-GI appears to be currently restricted to the clade ST313.The horizontal transfer of S. Typhimurium sequence type and is globally predominating st313-td among the two clonal groups. These results reveal that strains harbouring the gene are not restricted to the clonal group ST313 and sub-Saharan Africa. Recently, it has been suggested that birds might have been a vehicle or source of S. Typhimurium ST313 While ST313 has been specifically localized to the sub-Saharan Africa, ST19 is the most commonly identified st313-td was not detected in any S. Typhimurium strain of food or animal origin. Thus, in S. Typhimurium, the apparently restricted presence of st313-td in human isolates suggests a potential host specificity for this gene as suggested for its carrier, S. Typhimurium ST313. In a previous work, the authors showed reduced genome in the S. Typhimurium ST313 strain D23580, in comparison to gastroenteritis strains, a finding consistent with what might be expected of strains undergoing host adaptation Interestingly, st313-td mutant displayed diminished survival within J774 macrophages and was attenuated in virulence in a systemic murine infection. Thus, this work adds another chapter to understanding the virulence in S. Typhimurium but also highlights the need for a closer monitoring of this sequence type to control it and to avoid a further spread. Further work should be carried out for a deepest characterization of the genomic island and its possible essentiality in strains of ST313. Apart from immunity studies, prevention of NTS bacteraemia also requires genetic studies analysing the diversity of the circulating invasive strains. These studies contribute to investigate the epidemiology and transmission of this African pathogen, allow a better understanding of the pathogenesis of the NTS disease and represent key approaches for the developing of a much needed vaccine as there are limited antibiotic choices against NTS infections.We demonstrated that a Figure S1SNP tree showing the divergence/relatedness among the strains representing the identified ST types carrying st313-td. The reference strain S. Typhimurium D23580 and the ST313 isolate used in the present study 02–03/002 are highlighted in bold. Countries of origin and ST types are also indicated.(TIF)Click here for additional data file.Table S1Primers designed and used in this work.(DOCX)Click here for additional data file.Table S2Salmonella Typhimurium D23580 available at NCBI (accession no. FN424405.1). Uniprot database was used to determine the function of the encoded proteins.Characteristics of the proteins encoded by the ST313-GI. Sequence data (ORFs) extracted from the genome of the (DOCX)Click here for additional data file.


The results indicated that podophyllotoxin was capable of inhibiting growth and inducing the apoptosis of SGC-7901 cells in a dose-dependent manner, causing cell cycle arrest at the G2/M phase. After 48 h of treatment, the apoptotic morphology of SGC-7901 cells was clear, exhibiting cell protuberance, concentrated cytoplasms and apoptotic bodies. Following 24 h of treatment, the MMP of the SGC-7901 cells decreased. In addition, after 48 h, the expression of cyt-c was shown to be upregulated, while the expression levels of pro-caspase-9 and pro-caspase-3 in the SGC-7901 cells were shown to be downregulated. In conclusion, apoptosis can be induced in SGC-7901 cells by podophyllotoxin, potentially via a mitochondrial pathway, indicating that podophyllotoxin may be a potent agent for cancer treatment.Numerous studies have demonstrated that podophyllotoxin and its derivatives exhibit antitumor effects. The aim of the present study was to investigate SGC-7901 cell apoptosis and the underlying mechanism induced by podophyllotoxin. SGC-7901 cells were treated with varying concentrations of podophyllotoxin. MTT assays and flow cytometry were used to evaluate the effects of podophyllotoxin on the proliferation and apoptosis of SGC-7901 cells, while fluorescence inverted microscopy was used to observe the morphology of SGC-7901 cells that had been dyed with Hoechst 33258. In addition, laser scanning confocal microscopy was used to analyze the mitochondrial membrane potential (MMP) of SGC-7901 cells dyed with Rhodamine 123. Western blotting was performed to analyze the expression levels of cytochrome  Gastric cancer (GC) is one of the most common cancers worldwide, particularly in East Asia and East Europe. Furthermore, the incidence and mortality rates of GC are high, accounting for ~1,000,000 mortalities annually. Therefore, GC is a significant problem in terms of global health ,3.Podophyllum plant, with a molecular formula of C22H22O8 and a molecular weight of 414 Da. This compound and its derivatives have great significance as antineoplastic drugs and antiviral agents due to the biological activities that they exhibit. Podophyllotoxin and its derivatives are currently used in chemotherapy for various cancer types, including cervical carcinoma, osteosarcoma, nasopharyngeal carcinoma, colon cancer, breast cancer, prostate cancer, testicular carcinoma, small cell lung cancer and lymphoma , Hoechst 33258 and Rodamine 123 were all purchased from Sigma-Aldrich . Hydroxycamptothecin (HCPT) was purchased from Harbin Shengtai Pharmaceutical Co., Ltd. ; RPMI 1640 culture medium was purchased from Thermo Fisher Scientific Inc. ; fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.  and trypsin was purchased from Gibco-BRL . Additionally, mouse anti-human β-actin, cytochrome c (cyt-c), caspase-9 and caspase-3 polyclonal antibodies, alkaline phosphatase (AP)-conjugated goat anti mouse polyclonal antibody, SDS-PAGE sample loading buffer, blocking buffer, TBST, buffer5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT) alkaline phosphatase color development kit ; detergent-compatible protein assay kit .2 incubator was purchased from New Brunswick Scientific .The CKX 41 inverted fluorescence microscope was purchased from Olympus  and the mini electrophoresis meter and microplate reader were purchased from Bio-Rad . The EPICS XL flow cytometer was purchased from Beckman Coulter ; the SP2 laser confocal scanning microscope was purchased from Leica  and the CO-150 CO2.The human GC SGC-7901 cell line was provided by the Center of Research and Development on Life Sciences and Environmental Sciences of Harbin University of Commerce . SGC-7901 cells were grown in RPMI 1640 culture medium containing 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO5 cells/ml was achieved. In a 96-well plate, 100 μl cell suspension per well was cultured for 24 h. The cells were subsequently incubated with varying concentrations of podophyllotoxin for a further 72 h; 12 wells were used per concentration. Cells incubated with only 100 μl RPMI 1640 culture medium served as a control group. MTT was dissolved in phosphate-buffered saline (PBS) to provide a solution with a final concentration of 0.5 mg/ml. After 72 h, the cell suspension was discarded and 200 μl MTT solution (0.5 mg/ml) was added to each well and incubated at 37°C for 4 h. All media were removed and 150 μl dimethyl sulfoxide was added to each well in order to dissolve the purple formazan crystals. The plate was agitated for 3 min and the spectrophotometric absorbance at 570 nm was measured using a microplate reader. The inhibitory rate and IC50 was calculated.Exponentially growing cells were washed, digested with trypsin and suspended in RPMI 1640 medium until a concentration of 1×104/ml; 1 ml) were planted in 24-well plates and cultured for 24 h with three wells per group. Subsequently, the cells were treated with various drug concentrations for 48 h. The cells were harvested via trypsinization, washed twice with cold PBS and fixed in 70% cold ethanol for 12 h at 4°C. The fixation fluid was then discarded and the cells were washed with PBS twice. PI was added to the samples for 30 min for staining and the samples were measured by flow cytometry with an excitation wavelength of 488 nm.Cells  were planted in 6-well plates, cultured for 24 h and treated with varying drug concentrations for 48 h. Cells were harvested via trypsinization, washed twice with cold PBS and fixed in 4% paraformaldehyde for 30 min at 4°C. The fixation fluid was then discarded and the cells were washed with PBS twice. Hoechst 33258 was added for 20 min, after which the dye was discarded and the cells were washed twice with PBS. The cells were observed using an inverted fluorescence microscope.Cells  were planted in 6-well plates, cultured for 24 h and treated with various drug concentrations for 24 h. Cells were harvested via trypsinization, washed twice with cold PBS and loaded with Rhodamine 123 (5 μg/ml) for 30 min at 37°C. The fluorescence intensity of the MMP was observed using laser scanning confocal microscopy.Cells  were planted in 6-well-plates, cultured for 24 h and subsequently treated with varying concentrations of podophyllotoxin for 48 h. Cytoplasm extracts were prepared with 150 μl cell lysis buffer on ice for 30 min. Following centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was collected and the protein concentration was quantified using the detergent-compatible protein assay kit. Proteins were mixed with SDS-PAGE sample loading buffer (Beyotime Institute of Biotechnology). In total, a 40-μg sample of protein was separated in a 10% polyacrylamide gel and blotted on a nitrocellulose membrane. The blots were blocked with blocking buffer for 2 h at room temperature and incubated with anti-cyt-c, anti-caspase-9 and anti-caspase-3 antibodies for 12 h at 4°C. Subsequently, the nitrocellulose membranes were washed with TBST buffer three times and incubated with AP-conjugated goat anti-mouse antibody in blocking buffer for 2 h at R.T. Then the nitrocellulose membranes were washed with TBST buffer three times and detected by BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology). The bands were then visualized and quantified using the Gel Doc XR imaging system .Cells . P<0.05 was considered to indicate a statistically significant difference.50 was 26.30 μmol/l . The IC0 μmol/l .0/G1 and S phases decreased, while the number of cells in the G2/M phase increased. The apoptotic peaks appeared gradually in a concentration-dependent manner, and the apoptosis rates were 11.06, 20.39 and 33.67% in the 13.15, 26.30 and 52.60 μmol/l podophyllotoxin groups, respectively, as shown in Following treatment with varying concentrations of podophyllotoxin for 48 h, the cell cycle changed and the number of cells in the GUnder an inverted fluorescence microscope, the cells in the control group were observed to grow normally and the fluorescence in the cell nuclei was uniform. Following treatment with various concentrations of podophyllotoxin for 48 h, a high proportion of cells exhibited apoptosis-like changes, including cell detachment, cytoplasmic condensation and the appearance of apoptotic bodies, in a concentration-dependent manner .Following treatment with various concentrations of podophyllotoxin for 24 h, the fluorescence intensity of Rhodamine 123 in cells decreased, indicating that the level of the MMP had decreased. With increasing drug concentrations, the level of the MMP decreased accordingly and statistically significant differences were observed when compared with the control group (P<0.05), as shown in c increased, while the expression levels of pro-caspase-9 and pro-caspase-3 decreased in a concentration-dependent manner. Statistically significant differences were observed when compared with the control group (P<0.05), as shown in Following treatment with various concentrations of podophyllotoxin for 24 h, the expression levels of apoptosis-related proteins in SGC-7901 cells changed. The expression level of cyt-Apoptosis is a rigorous, active and orderly process of cell death that is regulated by numerous genes in order to maintain the stability of the intracellular environment. The mitochondrial pathway is one of three major apoptotic pathways. This pathway involves the induction of apoptosis factors, the life-or-death apoptosis switch, the start molecules of apoptosis (cyt-c) and the effector molecules of apoptosis, including caspase-9 and caspase-3.A change in the permeability of the mitochondrial membrane is the most significant event in apoptosis and this permeability is controlled by the mitochondrial permeability transition pore (MPTP), which is the key to the apoptotic mitochondrial pathway . The MMPc occurs in the early stages of cell apoptosis (c is released into the cytoplasm through mitochondrial outer membrane permeabilization, which is regulated by MPTP or members of the Bcl-2 family. The release of cyt-c results in the activation of caspases. Caspases are a type of proenzyme that, under normal conditions, contain no reactive site. In caspase-dependent mitochondrial pathways, cyt-c is released from the mitochondria and interacts with aminophospholipid transferase and apoptotic peptidase activating factor 1, subsequently converting these molecules to polymers and promoting their interaction with caspase-9 to form apoptotic bodies (c is capable of activating caspase-9 by hydrolyzing its proenzyme, and the activated caspase-9 further activates caspase-3, leading to cell apoptosis (c from the mitochondrion into the cytoplasm, which decreases the expression levels of pro-caspase-9 and pro-caspase-3, leading to the caspase cascade with the formation of a caspase-dependent pathway. Therefore, apoptosis is induced in SGC-7901 cells via the mitochondrial pathway.The release of cyt-poptosis . Cyt-c ic bodies . Cyt-c ipoptosis . The resIn conclusion, podophyllotoxin induces apoptosis in the human GC SGC-7901 cell line through the mitochondrial pathway.


Negative interpretation is thought to be crucial in the development and maintenance of depression. Recently developed cognitive bias modification paradigms, intending to change these biases towards a more optimistic interpretation tendency (CBM-I), seem to offer new promising implications for cognitive therapy innovation. This study aimed to increase our knowledge of the underlying mechanisms of action of imagery-based CBM-I in the context of depressed mood. We therefore compared the efficacy of CBM-I requiring participants to imagine standardized positive resolutions to a novel, more active training version that required participants to generate the positive interpretations themselves. Fifty-four participants were randomly allocated to (1) standardized CBM-I, (2) self-generation CBM-I or (3) a control group. Outcome measures included self-report mood measures and a depression-related interpretation bias measure. Both positive training variants significantly increased the tendency to interpret fresh ambiguous material in an optimistic manner. However, only the standardized imagery CBM-I paradigm positively influenced mood. How we disambiguate or make sense of these stimuli is important for how we further respond to the world. To interpret one's dialogue partner's yawning as a sign of boredom can obviously result in a different, even opposite emotion than attributing it to a simple lack of oxygen in the room. There is empirical consensus that negatively biased interpretation, defined as the tendency to consistently interpret ambiguous stimuli in a negative manner, is associated with dysphoria and depression and might play a key role in the development of disorders e.g. .Cognitive bias modification paradigms CBM;  are compAlthough CBM-I has initially been developed for the training of anxiety-related bias, positive benefits of interpretation modification training could also be reported in the context of dysphoria and depression by a number of studies . PositivA line of research e.g.  has beenway.” . While participants in the passive group were presented with the entire passage, individuals in the active group were presented with only a fragment (one or more letters missing) of the final word (“….completely lost your w--.”) and therefore had to actively resolve the meaning by themselves (only one possible completion). Active selection of meaning was shown to be superior in modifying later emotional responses in a training-congruent way to images of new emotionally ambiguous descriptions presented after training than mere passive exposure. Research in the context of anxiety-related interpretation bias has found active selection of meaning during the training to be critical for modifying subsequent emotional responses to new ambiguous stimuli . In thisThe self-perception theory  suggestsThe aim of this study was to develop a new, more active variant of CBM-I and test its impact on positive mood and interpretation bias in comparison to a control group as well as imagery CBM-I. The purpose of this comparison was to further our knowledge about the mechanisms of action underlying successful CBM-I and to optimize its ingredients to enhance its future therapeutic potential. The positive imagery CBM-I can be described as a standardized guided imagery training that provides participants with auditorily presented descriptions of ambiguous virtual everyday life situations that are consistently combined with positive resolutions e.g., . The new22.1M=22.0, SD=2.9). They were recruited via online advertisements at the website of the Technische Universität Dresden. The majority of them were undergraduate students. One third of the participants (29.6%) in this study showed BDI-scores above the clinical cut-off score with 22.2% reporting symptoms of mild depression (BDI-scores: 14–19) and 7.5% reporting symptoms of moderate depression (BDI-scores: 20–28). Reimbursement for participation consisted of either a small participation fee (5€) or course credits.54 participants  took part in the study . This part was also ambiguous in that it allowed either a positive or a negative outcome. Each statement lasted about 5–10 s. Scenarios in all groups were recorded using the same female voice and presented stereo-phonically through headphones.Participants were randomly assigned to (1) standardized CBM-I-, (2) self-generation CBM-I, or (3) a control group (CG). Using the CBM-I paradigm described by The second part of the scenarios differed between training conditions. In the standardized CBM-I condition, the auditory description continued directly on with the positive resolution (e.g. “ The pictures are much better than you expected.”). Participants had to vividly imagine the complete scenario including its positive resolution (no time limit was given) and then had to press a “continue”—button to start the next scenario. Participants in the condition self-generation CBM-I were presented the initial part of the scenario. They were instructed (before training) to vividly imagine the auditorily presented scenario and to resolve its ambiguity by imagining a positive completion by themselves. After the imagery part (no time limit was given) participants had to press a “start”-button and were asked to describe their actively generated positive resolution by speaking a few words into a microphone . Individuals had to press a “continue”—button to start the next scenario. Participants in the control group were merely presented the first part of the scenario with no resolution. They had to vividly imagine each scenario and had to press a “continue”—button to start the next scenario.2.2.4Following previous studies e.g. , in orde2.2.5During the training, participants gave ratings at the end of each 10th scenario regarding the previously presented scenario. Participants were asked to rate (a) to what degree they had been able to vividly imagine the scenario (vividness) (b) as how realistic they had experienced the presented scenario (authenticity) and (c) as how pleasant they had perceived the presented scenario (pleasantness). Responses were rated on a 9-point scale, ranging from 1  to 9 (extremely).2.2.6Furthermore, participants were given five memory questions at the end of the session (e.g. “What present did you buy for your partner (in one of the presented scenarios)?”). These questions were part of the cover story and additionally served as a filter to exclude participants with insufficient ability to concentrate on the training. Since imagery has been associated with enhanced learning and memory effects e.g. , we expe2.3experimental phase). After having given informed consent and being randomly assigned to one of the three training-conditions, participants completed BDI-II, STAI-T, PANAS, as well as the pre-test of the AST-D-II. After the experimental phase (CBM-I), they again completed the PANAS. This state questionnaire was given for a third time after the 10 min filler task. Participants then completed the post-test AST-D-II. They completed the manipulation check ratings  during the training. At the end of the study, they answered five memory questions and were asked about their expectancy beliefs regarding the aim of the study.In order to decrease expectancy as well as demand effects, participants were provided with a cover story. They were told that the purpose of the study was to examine the association between memory effects and spatial representations, and that they would have to answer several questions concerning their mental images after an imagery-task (33.1X2(2)=0.20, p=.90, mean age =0.20, p=.82), depressive symptoms =2.20, p=.12), trait anxiety =1.30, p=.28), mood =0.45, p=.64; PANAS-negative; F=0.94, p=.40), nor interpretation tendencies =0.40, p=.67).Questionnaire scores of all baseline measures are depicted in 3.2Negative subscale: There was no significant main effect of the factors time, F=0.09, p=.76, or group, F=1.15, p=.32, nor a significant interaction, all F=0.54, p=.58, indicating that none of the trainings lead to a change in negative mood. Positive subscale: There was no main effect of group, F=1.27, p=.29, but a significant effect of time, F=10.56, p<.01, η2p=.17, as well as a significant interaction between time and group, F=4.05, p=.02, η2p=.14, indicating differential changes in positive mood in the three groups during training. Additional paired-samples t-tests comparing pre- and post-CBM mood scores, separated for the three CBM groups, indicated a significant increase of positive mood during standardized CBM-I, t(17)=4.32, p<.01, whereas no change was found in self-generation CBM-I, t(17)=1.17, p=.26, and the control group, t(17)=0.35, p=.73. There was a trend level difference between the three groups' levels of positive affect post-CBM, F=2.43, p=.09. In summary, our analysis revealed no changes in negative mood in any of the training conditions, but an increase of positive mood in the standardized CBM-I used in earlier studies.Two 2 x 3 repeated measures ANOVAs with the within-factor time (pre- vs. post training) and the between-factor group  were calculated using the pre- and post-test scores of the PANAS as the dependent variables, separated for the positive and the negative PANAS subscale , Fig. 1.3.2.1F=2.02, p=.14, but a significant effect of time, F=57.36, p<.01, η2p=.53, and a significant interaction between time and group, F=12.16, p<.01, η2p=.32, indicating differential changes in mood in the three groups during the filler task. Follow-up paired-samples t-tests, comparing pre- and post-filler positive mood, indicated a significant increase of positive mood in the control group, t(17)=9.22, p<.01 and self-generation CBM-I, t(17)=3.69, p<.01, whereas no change was found in standardized CBM-I, t(17)=1.00, p=.33. In summary, our analysis revealed no mood transition in standardized CBM-I, but mood changes in self-generation CBM-I as well as in the control group.To assess whether the mood improvement during training in the standardized CBM-I group survives a neutral filler task or is rather transient, we ran an additional 2×3 repeated measures ANOVA with the within-factor time (pre- vs. post filler) and the between-factor group, using the pre- and post-filler task scores of the positive subscale of the PANAS as the dependent variable. The was no significant main effect of group, 3.3F<1.65, both p>.20. This indicates that the groups were comparable prior to the administration of the bias tasks.Two one-way ANOVAs, separated for the negative and the positive subscale of the PANAS, were run for the scores recorded after the filler task to compare mood across the three CBM groups. No significant differences between the three training groups were found, neither for negative nor positive affect scores, both 3.4F=0.30, p=.74, but a significant effect of time, F=16.68, p<.01, η2p=.25, and a significant interaction between time and group, F=5.23, p<.01, η2p=.17, indicating differential changes in interpretation bias in the three groups during training. Additional paired samples t-tests separately conducted for each of the three groups indicated a significant increase of positive interpretation bias during training in the standardized CBM-I group, t(17)=3.69, p<.01, and the self-generation CBM-I group, t(17)=3.33, p<.01, whereas no change was found in the CG, t(17)=0.13, p=.90. Independent-samples t-tests suggested no difference between the training effect scores of standardized CBM-I and self-generation CBM-I, t(34)=1.08, p=.29, indicating these two trainings as equally effective in changing interpretation style towards a more positive direction. There was no significant difference in AST-D-II scores post-training between the three groups, F=1.98, p=.15. In summary, participants in the positive training conditions, but not in the control group, showed a similar increase of positive interpretation as measured with the AST-D-II.A 2×3 repeated measures ANOVA with the within-factor time (pre- vs. post training) and the between factor group  with the pre- and post-training scores of the AST-D-II as the dependent variables was run , Fig. 2.3.5F<1.82, p>.17. However, a significant difference was found regarding their pleasantness ratings, F=1.82, p<.01, η2p=.54. Subsequent post-hoc pair wise comparisons (Bonferroni) revealed that the control group rated the scenarios as significantly less pleasant than did the two positive training conditions (both p<.01). None of the participants correctly guessed the purpose of the study when asked at the end.Using one-way ANOVAs, the groups did not differ regarding their ratings of the vividness and the authenticity of the situation samples, nor their imagery-related memory performance, all 4The goal of this study was to design and evaluate a new self-generation variant of imagery CBM-I and test its effectiveness in comparison to a control group as well as standard or guided imagery CBM-I. The purpose of this comparison was to increase the knowledge about the mechanisms of action behind this cognitive bias modification procedure. Our self-generation version of CBM-I increased positive interpretation, whereas no such change was seen in the control condition. We had assumed that self-generation CBM-I would be more effective than the standardized variant, based on the principles of self-perception as well as prior findings by How can these findings be explained? We had suggested that the self-generation of positive resolutions  as a more active and overt behaviour would lead – according to the self-perception theory – to greater changes in interpretation bias as well as mood. We had expected that observing themselves generating the (positive) resolutions for the scenarios would facilitate participants internalising these resolutions as their own expectations for the outcomes of the imagined situations. From a self-perception theory perspective, the lack of additional effect on modification of interpretation bias of this self-generation could perhaps be explained by the idea that mental imagery from a field perspective is like an “as-if” experience cf. . That isInterestingly, the finding of similar effects on interpretation after self-generated and standardized CBM-I could be seen as also contrasting with previous research, showing stronger effects of “active” compared to “passive” procedures in modifying later responses to new emotionally ambiguous descriptions . HoweverWhy was there an unexpected lack of mood improvement during our self-generation CBM-I training as opposed to the significant increase of positive mood in standardized CBM-I, that even remained stable over a filler task? Perhaps the generation of positive outcomes may (at least in this study) place a higher demand on concentration and performance, reducing any potential increase in positive affect. The requirement to concentrate on self-generating an ending may have also disrupted participants' absorption in the positive imagery, reducing its emotional impact, or resulted in more evaluative post-processing of the image via the requirement to monitor the valence of the ending generated. That is, having to “think about” the positive endings may have distracted from the “as if” processing that characterizes mental imagery, and promoted more comparative, evaluative processing cf. . The twoWhat might be the significance of the greater increase in positive mood seen in the standardized imagery CBM-I? Although the main aim of the CBM-I paradigm may be to train more positive interpretation, if the training sessions also boost positive mood then this may have significant implications for future clinical application in depression. Positive affect may be an important determinant of clinical outcome in depression, with lack of positive affect predicting poorer prognosis e.g. , and earOn the basis of prior research e.g.  and suppA few limitations of the study need to be highlighted. First, our assessments relied on self-report, involving the risk of response bias. However, as none of the individuals in our sample correctly guessed the study purpose, the influence of demand or expectancy effects on results is rather unlikely. Furthermore, preliminary studies using more involuntary, psycho-physiological measures of interpretation provide further evidence for the association between interpretation bias and depression e.g. . NeverthIn summary, the current study adds further support to the potential therapeutic significance of guided CBM-I-based imagery techniques and evaluated a new, self-generation variant of CBM-I.Although some of the authors are supported by funding from sources Elsevier has an agreement with, this research has been supported by funds other than these.None of the authors have any conflict of interest to report.